Experimental free energy surface reconstruction from single-molecule force spectroscopy using Jarzynski's equality

Area of Science:

• Single-molecule biophysics
• Statistical mechanics
• Molecular dynamics

Background:

• Understanding protein folding and unfolding is crucial in molecular biology.
• Characterizing the free energy landscape of proteins provides insights into their stability and function.
• Traditional methods often struggle to accurately measure energy landscapes under non-equilibrium conditions.

Purpose of the Study:

• To reconstruct the free energy surface of the titin I27 domain during stretching and unfolding.
• To apply Jarzynski's equality to single-molecule force spectroscopy experiments.
• To directly determine the unfolding free energy barrier from experimental data.

Main Methods:

• Utilizing atomic force microscopy (AFM) to manipulate and unfold individual titin I27 domain molecules.
• Applying Jarzynski's equality, an exact formula relating non-equilibrium work fluctuations to free energy.
• Analyzing experimental data to reconstruct the free energy surface and identify the activation energy barrier.

Main Results:

• Successfully reconstructed the free energy surface for both stretching and unfolding of the titin I27 domain.
• Demonstrated the direct measurement of the unfolding free energy barrier for the first time using experimental data.
• Validated the applicability of Jarzynski's equality in analyzing non-equilibrium single-molecule experiments.

Conclusions:

• Jarzynski's equality provides a powerful tool for analyzing non-equilibrium single-molecule experiments.
• This method enables the determination of free energy surfaces for molecular systems where only non-equilibrium work can be measured.
• The study offers a novel approach to quantifying protein stability and dynamics at the single-molecule level.